G Protein-Coupled Receptors (GPCRs) are integral membrane proteins that regulate multiple and diverse cellular signaling pathways to control essential physiological functions in humans. A significant unsolved problem in signaling biochemistry is to understand the mechanism by which G proteins are activated by GPCRs. Elucidation of the three-dimensional structure of the complex between a GPCR in its agonist activiated state with the G protein heterotrimer upon which it acts, is essential to the solution of this problem. Because pharmacological intervention in a variety of disease states is directed toward specific modulation of GPCRs, knowledge of their three-dimensional structures in their active signaling states will provide important information to guide the design of receptor agonists and inverse-agonists. The goal of this proposal is to use fusion proteins in which GPCRs are linked to G protein alpha subunits as scaffolds for in vivo reconstitution, over-expression and purification of GPCR-G protein complexes in their active, signaling state. Specific vector design and mutagenesis strategies will be implemented to optimize expression of stable complexes in insect cell lines and enable their efficient purification. Methods to stabilize GPCR-G protein complexes, including development of specific detergent/lipid combinations and FAB fragments of monoclonal antibodies that recognize the native structure of the complexes, will be developed. Conditions will the defined that lead to formation of crystals of GPCR-G proteins or their complexes with monoclonal FAB fragments, suitable for determination of their three-dimensional structures. Because its biochemical and pharmacological properties are well characterized, beta 2 adrenergic receptor-Gs fusion protein will serve as the prototype for these studies. At the same time, two other receptor-G protein fusions will be developed to exploit variations in the pharrmacological and biochemical properties of these receptors, their ligands and G protein signaling partners that may provide specific experimental advantages.